title: Sonic hedgehog signaling protects human hepatocellular carcinoma cells against ionizing radiation in an autocrine manner.
abstract: OBJECTIVE
  Sonic hedgehog (Shh) signaling is critical to embryogenesis and resistance to chemotherapy. We aimed to examine the role of Shh signaling in the response to radiation of human hepatocellular carcinoma (HCC) cells.
  
  
  METHODS
  Response to ionizing radiation therapy (RT) was evaluated by clonogenic assay. Quantitative RT-polymerase chain reaction for patched-1 (PTCH-1) expression was performed. Cytosolic accumulation of Shh and nuclear translocation of Gli-1 were assessed by immunofluorescence. Gli-1 knockdown was done by RNA interference (RNAi). Immunoprecipitation was performed to detect Shh ligand in conditioned medium. Immunofluorescent stain for γ-H2AX was used as an index of DNA double strand breaks (DSB). Expression of proteins related to DNA damage repair was assessed by Western blotting.
  
  
  RESULTS
  We found that Shh ligand could protect human HCC HA22T and Sk-Hep1 cells against RT. In HA22T cells, Shh ligand activated the Shh signaling with upregulation of Shh, PTCH-1, and Gli-1 expression. The nuclear translocation of Gli-1 further supports the activation of Gli-1. The radioprotection by Shh ligand was partly blocked by Shh antibody neutralization and was abolished by Gli-1 RNAi, suggesting a critical role of Shh signaling in radiation resistance. Furthermore, we noted that soluble factors secreted into conditioned medium, either constitutively or responding to radiation, by HA22T or Sk-Hep1 cells protected subsequent culturing cells against RT. Immunoprecipitation shows the presence of Shh peptide in conditioned medium. Intriguingly, antibody neutralization of Shh ligand or knockdown of Gli-1 reversed the radioprotective effect of conditioned medium. Furthermore, Shh ligand reduced the RT-induced phosphorylation of checkpoint kinase 1 and impaired the repair of DNA DSB.
  
  
  CONCLUSIONS
  Activation of Shh signaling protects HCC cells against ionizing radiation in an autocrine manner. Impairment of DNA damage repair might involve mechanism of Shh-induced radioresistance. Targeting Shh signaling pathway may be a novel strategy to enhance the radioresponse of human HCC cells.
authors.affiliation: Department of Radiation Oncology, Mackay Memorial Hospital, Taipei, Taiwan.
authors.forename: Yu-Jen
authors.initials: YJ
authors.lastname: Chen
authors.forename: Chin-Ping
authors.initials: CP
authors.lastname: Lin
authors.forename: Ming-Ling
authors.initials: ML
authors.lastname: Hsu
authors.forename: Hui-Ru
authors.initials: HR
authors.lastname: Shieh
authors.forename: Nicholas K
authors.initials: NK
authors.lastname: Chao
authors.forename: K S Clifford
authors.initials: KS
authors.lastname: Chao
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000532:radiotherapy*; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D017077:Culture Media, Conditioned / Q000737:chemistry; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D053823:Hedgehog Proteins / Q000032:analysis / Q000502:physiology*; D006657:Histones / Q000032:analysis; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000532:radiotherapy*; D009363:Neoplasm Proteins / Q000502:physiology*; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology; D011836:Radiation Tolerance / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000071676:Zinc Finger Protein GLI1
chemical_list: D017077:Culture Media, Conditioned; C400551:GLI1 protein, human; C468783:H2AX protein, human; D053823:Hedgehog Proteins; D006657:Histones; D009363:Neoplasm Proteins; C000606236:PTCH1 protein, human; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D011956:Receptors, Cell Surface; C509239:SHH protein, human; D014157:Transcription Factors; D000071676:Zinc Finger Protein GLI1; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1